The goal of the proposed research is a complete description of mRNA synthesis at the near atomic level. RNA chain elongation, once regarded as a matter of monotonous ribonucleotide addition, is now known to comprise a complex interplay of RNA polymerase states and interacting protein factors. We have previously determined the X-ray structures of pre- and post-translocated states, leading to the trigger loop hypothesis for specificity and catalysis in the post-translocated state. We now propose a combination of genetic and biochemical studies, as well as extension to higher resolution, to evaluate this hypothesis and fully illuminate the catalytic mechanism. We will also determine the remaining RNA polymerase structures, those of paused and arrested backtracked states. Structures of cocrystals with auxiliary protein factors will reveal the basis for transitions between states, for proofreading the transcript in the paused state, for recovery from the arrested state, and for transcriptional regulation. Aims for the next project period are as follows: 1. Determine structures of transcribing complexes containing RNA polymerase mutations and nucleotide analogs. Polymerase mutants will be derived from a comprehensive genetic screen, which identifies amino acid residues important for transcription elongation in vivo. Nucleotide analogs will vary in size, shape, and hydrogen-bonding capability. Resolution will be extended with better crystals, data collection on microfocus beamlines, and improved data processing. The results will test the trigger loop hypothesis and give detailed insight into the transcription mechanism. 2. Determine structures of backtracked complexes. Short lengths of backtracked RNA will give rise to paused complexes, and longer backtracked RNAs will recreate the arrested state. The results will test hypotheses for interaction of the longer RNAs with the polymerase and for the control of backtracking. 3. Determine structures of cocrystals with TFIIS, Spt4/5, and Ubc5/Rsp5. TFIIS provokes the cleavage of backtracked RNAs, resulting in proofreading of paused complexes and recovery from the arrested state. Spt4/5 plays a key role in the regulation of transcription through promoter-proximal pausing. Ubc5/Rsp5 couple arrest to the ubiquitylation and destruction of RNA polymerase II.